In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function. IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

Mason-Pfizer monkey virus (M-PMV) proteinase, released by the autocatalytic cleavage of Gag-Pro and Gag-Pro-Pol polypeptide precursors, catalyzes the processing of viral precursors to yield the structural proteins and enzymes of the virion. In retroviruses, usually only one proteolytically active form of proteinase exists. Here, we describe an unusual feature of M-PMV, the existence of three active forms of a retroviral proteinase with molecular masses of 17, 13, and 12 kDa as determined by mass spectroscopy. These forms arise in vitro by self-processing of a 26-kDa proteinase precursor. We have developed a process for isolation of each truncated product and demonstrate that all three forms display proteolytic activity. Amino acid analyses, as well as the determination of N- and C-terminal sequences, revealed that the N-termini of all three forms are identical, confirming that in vitro autoprocessing of the 17-kDa form occurs at the C-terminus to yield the truncated forms. The 17-kDa form and the newly described 13-kDa form of proteinase were identified in virions collected from the rhesus monkey CMMT cell line chronically infected with M-PMV, confirming that multiple forms exist in vivo.

Gag polyprotein precursors play an essential role in the assembly of the HIV particle by polymerizing into a spherical shell at the plasma membrane. In order to define the domains within Gag responsible for this homotypic interaction, we have coupled the technology of the yeast two-hybrid system with the technology of a gene-based, semirandom library. By this method, we have identified a minimal region of Gag capable of efficient self-interaction. This region consists of the N-terminal portion of the nucleocapsid protein (NC), including the first zinc finger and the previously described interaction, or I, domain. In parallel with this randomized approach, individual HIV Gag domains, and combinations of these domains, were tested for potential homotypic and heterotypic interactions in the yeast two-hybrid system. Consistent with the results from the semirandom library screen, only combinations of species containing NC were strongly interacting.

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